title: Evaluation of protein extraction protocols for 2DE in marine ecotoxicoproteomics.
abstract: In ecotoxicoproteomics, an accurate and reproducible extraction of proteins is a critical step for 2DE analysis and further protein identification using MS. The criteria for the assessment of protein extraction quality include protein yield, protein spots resolved in a 2DE gel, matched protein spots in replicate gels, reproducibility, and compatibility with MS. In this work, we evaluated three protein extraction systems, straightforward lysis buffer, trichloroacetic acid-acetone, and TRIzol reagent with some modifications, for the protein extraction from three animal species including mussel Mytilus galloprovincialis, flounder Paralichthys olivaceus, and polychaete Nereis diversicolor used in marine ecotoxicology. Our results indicated that these methods could extract significantly different protein profiles. The method using TRIzol reagent resulted in the most matched protein spots resolved in four replicate 2DE gels and highest reproducibilities for the gill of M. galloprovincialis and liver of P. olivaceus. However, a modified trichloroacetic acid-acetone solvent system was best for the whole soft tissue of N. diversicolor. This work provides the fundamental information of the extraction quality of protein extraction protocols from different marine animals, which may facilitate the selection of a suitable protein extraction protocol for ecotoxicoproteomics.
authors.affiliation: Key Laboratory of Coastal Zone Environmental Processes, Yantai Institute of Coastal Zone Research (YIC), Chinese Academy of Sciences (CAS); Shandong Provincial Key Laboratory of Coastal Zone Environmental Processes, YICCAS, Yantai, P. R. China.
authors.forename: Huifeng
authors.initials: H
authors.lastname: Wu
authors.forename: Chenglong
authors.initials: C
authors.lastname: Ji
authors.forename: Lei
authors.initials: L
authors.lastname: Wei
authors.forename: Jianmin
authors.initials: J
authors.lastname: Zhao
mesh_terms: D000818:Animals; D059001:Aquatic Organisms / Q000737:chemistry*; D054750:Ecotoxicology / Q000379:methods*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005432:Flounder; D005880:Gills / Q000737:chemistry; D008099:Liver / Q000737:chemistry; D013058:Mass Spectrometry; D049878:Mytilus / Q000737:chemistry; D011077:Polychaeta / Q000737:chemistry; D025341:Principal Component Analysis; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D011506:Proteins
keywords: 2DE; Animal proteomics; Ecotoxicoproteomics; Protein extraction; Reproducibility